PROJECT SUMMARY PD1 and LAG3 play critical roles in regulating T cell tolerance and autoimmunity. Combined genetic deficiency of PD1 and LAG3 in mice results in lethal systemic autoimmunity, demonstrating PD1/LAG3 synergies in tolerance. The goal of Project 1 is to determine mechanisms by which PD1 and LAG3 control Treg and self- reactive CD4+ FoxP3? T cells in tolerance and autoimmunity. These are issues of fundamental and clinical significance, as PD1 and LAG3 are key mediators of T cell exhaustion and promising therapeutic targets, and there is a growing appreciation of immune-related adverse events in cancer patients treated with checkpoint blockade. A deeper understanding of how PD1 and LAG3 work together to mediate tolerance may enable optimal effective PD1 and LAG3 cancer therapies, while minimizing autoimmunity. In contrast to exacerbated EAE in Pdcd1?/? mice, our preliminary data show that mice lacking PD1 in Treg develop ameliorated EAE. Conversely, LAG3 deletion in Treg increased EAE severity, while combined PD1/LAG3 deletion in Treg reduced EAE. These findings highlight the need to determine how PD1/LAG3 signals are integrated in different cell types to understand how perturbing these pathways impacts protective Treg and pathogenic self-reactive CD4+ FoxP3? T cells. Based on these data, we hypothesize that synergy between PD1 and LAG3 operates by cellular and molecular mechanisms that differ in Treg and CD4+ FoxP3? T cells to control pathogenic and protective responses during EAE initiation and progression. We will test this hypothesis by clinical, cellular, and molecular analyses of a) novel inducible knockout mice where PD1 and/or LAG3 can be temporally deleted only in Treg cells or only in CD4+ Foxp3? T cells and b) mice given PD1 and/or LAG3 blocking antibodies. We will evaluate select targets from our studies to define mechanisms by which PD1/LAG3 coordinate to control cell fate and function. Aim 1: What cellular and molecular events are triggered in Treg and self-reactive CD4+ FoxP3? T cells by PD1/ LAG3 deletion or blockade during EAE onset? We hypothesize that PD1/LAG3 interactions have distinct effects on Treg vs. CD4+ FoxP3? T cells during EAE initiation. We will define how PD1 and/or LAG3 deletion affects self-reactive CD4+ Foxp3? T cell activation/differentiation and Treg activation/function, and integrated effects of LAG3/PD1 blockade. Aim 2: What cellular and molecular events are triggered in Treg and self-reactive CD4+ Foxp3? effector (Teff) cells by PD1/LAG3 deletion or blockade after EAE onset? We predict that PD1/LAG3 interactions may differ in initiation vs. effector phases of EAE, since PD1 can regulate T cell differentiation fates and effector responses. We will define how PD1 and/or LAG3 deletion impacts Teff and Treg cells and integrated effects of PD1/LAG3 blockade. Project 1 will collaborate with Projects 2 and 3 to compare effects of PD1/LAG3 disruption in tolerance and T cell exhaustion: CD4+ FoxP3? T cells in EAE vs. cancer (Project 2), and CD4+ Teff in EAE vs. CD8+ T cells in cancer (Project 2) and chronic infection (Project 3). Project 1 will interact with Core A to exchange data, Core B to obtain unique mouse strains, Core C for transcriptional analyses, and Core D for immunopathology studies.